The alarmones (p)ppGpp directly regulate translation initiation during entry into quiescence.

Many bacteria exist in a state of metabolic quiescence where energy consumption must be minimized so as to maximize available resources over a potentially extended period of time. As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for down-regulation during the transition from growth to quiescence. We observe that when Bacillus subtilis exits rapid growth, a subpopulation of cells emerges with very low protein synthetic activity. This phenotypic heterogeneity requires the production of the nucleotides (p)ppGpp, which we show are sufficient to inhibit protein synthesis in vivo. We then show that one of these molecules, ppGpp, inhibits protein synthesis by preventing the allosteric activation of the essential GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (p)ppGpp and IF2.

SIRT2 Affects Primary Cilia Formation by Regulating mTOR Signaling in Retinal Pigmented Epithelial Cells.

SIRT2, a member of the Class III HDAC family, participates in diverse cellular processes and regulates several pathological conditions. Although a few reports show that SIRT2 regulates the cell cycle, the causes and outcomes of SIRT2-dependent cell proliferation remain unclear. Here, we examined the effects of SIRT2 suppression in human RPE1 cells using siRNA targeting SIRT2, and AK-1, a SIRT2-specific inhibitor. The number of primary cilia in SIRT2-suppressed cells increased under serum-present conditions. Suppressing SIRT2 induced cell cycle arrest at G0/G1 phase by inactivating mammalian target of rapamycin (mTOR) signaling, possibly through mTORC1. Treatment with torin 1, an inhibitor of mTORC1/mTORC2, yielded results similar to those observed after SIRT2 suppression. However, SIRT2 suppression did not affect primary cilia formation or mTOR signaling following serum starvation. This suggests that SIRT2 acts as a critical sensor that links growth factor-dependent signal transduction and primary cilia formation by regulating the cell cycle.

Tat-Dependent Heterologous Secretion of Recombinant Tyrosinase by Pseudomonas fluorescens Is Aided by a Translationally Fused Caddie Protein.

Tyrosinase is a monooxygenase that catalyzes both the hydroxylation of p-hydroxyphenyl moieties to o-catechols and the oxidation of o-catechols to o-quinones. Apart from its critical functionality in melanogenesis and the synthesis of various neurotransmitters, this enzyme is also used in a variety of biotechnological applications, most notably mediating covalent cross-linking between polymers containing p-hydroxyphenyl groups, forming a hydrogel. Tyrosinases from the genus Streptomyces are usually secreted as a complex with their caddie protein. In this study, we report an increased secretion efficiency observed when the Streptomyces antibioticus tyrosinase gene melC2 was introduced into Pseudomonas fluorescens along with its caddie protein gene melC1, which has the DNA sequence for the Tat (twin-arginine translocation) signal.IMPORTANCE We observed that the S. antibioticus extracellular tyrosinase secretion level was even higher in its nonnatural translationally conjugated fusion protein form than in the natural complex of two separated polypeptides. The results of this study demonstrate that tyrosinase-expressing P. fluorescens can be a stable source of bacterial tyrosinase through exploiting the secretory machinery of P. fluorescens.

CCAR2/DBC1 and Hsp60 Positively Regulate Expression of Survivin in Neuroblastoma Cells.

CCAR2 (cell cycle and apoptosis regulator 2) controls a variety of cellular functions; however, its main function is to regulate cell survival and cell death in response to genotoxic and metabolic stresses. Recently, we reported that CCAR2 protects cells from apoptosis following mitochondrial stress, possibly by co-operating with Hsp60. However, it is not clear how CCAR2 and Hsp60 control cell survival and death. Here, we found that depleting CCAR2 and Hsp60 downregulated expression of survivin, a member of the inhibitor of apoptosis (IAP) family. Survivin expression in neuroblastoma tissues and human cancer cell lines correlated positively with expression of CCAR2 and Hsp60. Furthermore, high expression of CCAR2, Hsp60, and survivin was associated with poor survival of neuroblastoma patients. In summary, both CCAR2 and Hsp60 are required for expression of survivin, and both promote cancer cell survival, at least in part, by maintaining survivin expression. Therefore, CCAR2, Hsp60, and survivin are candidate tumor biomarkers and prognostic markers in neuroblastomas.

The Protein Phosphatase PPM1G Destabilizes HIF-1α Expression.

Hypoxia-inducible factors (HIFs) are key regulators of hypoxic responses, and their stability and transcriptional activity are controlled by several kinases. However, the regulation of HIF by protein phosphatases has not been thoroughly investigated. Here, we found that overexpression of Mg2+/Mn2+-dependent protein phosphatase 1 gamma (PPM1G), one of Ser/Thr protein phosphatases, downregulated protein expression of ectopic HIF-1α under normoxic or acute hypoxic conditions. In addition, the deficiency of PPM1G upregulated protein expression of endogenous HIF-1α under normoxic or acute oxidative stress conditions. PPM1G decreased expression of HIF-1α via the proteasomal pathway. PPM1G-mediated HIF-1α degradation was dependent on prolyl hydroxylase (PHD), but independent of von Hippel-Lindau (VHL). These data suggest that PPM1G is critical for the control of HIF-1α-dependent responses.

Zingerone Suppresses Tumor Development through Decreasing Cyclin D1 Expression and Inducing Mitotic Arrest.

Cancer cells undergo uncontrolled proliferation resulting from aberrant activity of various cell-cycle proteins. Therefore, despite recent advances in intensive chemotherapy, it is difficult to cure cancer completely. Recently, cell-cycle regulators became attractive targets in cancer therapy. Zingerone, a phenolic compound isolated from ginger, is a nontoxic and inexpensive compound with varied pharmacological activities. In this study, the therapeutic effect of zingerone as an anti-mitotic agent in human neuroblastoma cells was investigated. Following treatment of BE(2)-M17 cells with zingerone, we performed a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and colony-formation assay to evaluate cellular proliferation, in addition to immunofluorescence cytochemistry and flow cytometry to examine the mitotic cells. The association of gene expression with tumor stage and survival was analyzed. Furthermore, to examine the anti-cancer effect of zingerone, we applied a BALB/c mouse-tumor model using a BALB/c-derived adenocarcinoma cell line. In human neuroblastoma cells, zingerone inhibited cellular viability and survival. Moreover, the number of mitotic cells, particularly those in prometaphase, increased in zingerone-treated neuroblastoma cells. Regarding specific molecular mechanisms, zingerone decreased cyclin D1 expression and induced the cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1). The decrease in cyclin D1 and increase in histone H3 phosphorylated (p)-Ser10 were confirmed by immunohistochemistry in tumor tissues administered with zingerone. These results suggest that zingerone induces mitotic arrest followed by inhibition of growth of neuroblastoma cells. Collectively, zingerone may be a potential therapeutic drug for human cancers, including neuroblastoma.

A vector system for ABC transporter-mediated secretion and purification of recombinant proteins in Pseudomonas species.

Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species.

Structural basis of odor sensing by insect heteromeric odorant receptors.

Most insects, including human-targeting mosquitoes, detect odors through odorant-activated ion channel complexes consisting of a divergent odorant-binding subunit (OR) and a conserved co-receptor subunit (Orco). As a basis for understanding how odorants activate these heteromeric receptors, we report here cryo-electron microscopy structures of two different heteromeric odorant receptor complexes containing ORs from disease-vector mosquitos Aedes aegypti or Anopheles gambiae. These structures reveal an unexpected stoichiometry of one OR to three Orco subunits. Comparison of structures in odorant-bound and unbound states indicates that odorant binding to the sole OR subunit is sufficient to open the channel pore, suggesting a mechanism of OR activation and a conceptual framework for understanding evolution of insect odorant receptor sensitivity.

dDrop-Chip: disposable film-chip microfluidic device for real-time droplet feedback control.

A cost-effective, simple to use, and automated technique that can provide real-time feedback control for droplet generation is required to obtain droplets with high-throughput, stability, and uniformity. This study introduces a disposable droplet generation microfluidic device (dDrop-Chip) that can simultaneously control both droplet size and production rate in real time. The dDrop-Chip consists of a reusable sensing substrate and a disposable microchannel that can be assembled using vacuum pressure. It also integrates a droplet detector and a flow sensor on-chip, enabling real-time measurement and feedback control of droplet size and sample flow rate. The dDrop-Chip has the additional advantage of being disposable, which can prevent chemical and biological contamination, due to low manufacturing cost by the film-chip technique. We demonstrate benefits of the dDrop-Chip by controlling droplet size at a fixed sample flow rate and the production rate at a fixed droplet size using real-time feedback control. The experimental results show that the dDrop-Chip consistently generates monodisperse droplets with a length of 219.36 ± 0.08 µm (CV 0.036%) at a production rate of 32.38 ± 0.48 Hz using the feedback control, while without feedback control, there is a significant deviation in droplet length (224.18 ± 6.69 µm, CV 2.98%) and production rate (33.94 ± 1.72 Hz) despite the use of identical devices. Therefore, the dDrop-Chip is a reliable, cost-effective, and automated technique for generating droplets of controlled size and production rate in real time, making it suitable for various droplet-based applications.

Reducing sprint time with exosuit assistance in the real world.

Hip extension assistance with the aid of exosuits can reduce sprinting time.

Effect of hip abduction assistance on metabolic cost and balance during human walking.

The use of wearable robots to provide walking assistance has rapidly grown over the past decade, with notable advances made in robot design and control methods toward reducing physical effort while performing an activity. The reduction in walking effort has mainly been achieved by assisting forward progression in the sagittal plane. Human gait, however, is a complex movement that combines motions in three planes, not only the sagittal but also the transverse and frontal planes. In the frontal plane, the hip joint plays a key role in gait, including balance. However, wearable robots targeting this motion have rarely been investigated. In this study, we developed a hip abduction assistance wearable robot by formulating the hypothesis that assistance that mimics the biological hip abduction moment or power could reduce the metabolic cost of walking and affect the dynamic balance. We found that hip abduction assistance with a biological moment second peak mimic profile reduced the metabolic cost of walking by 11.6% compared with the normal walking condition. The assistance also influenced balance-related parameters, including the margin of stability. Hip abduction assistance influenced the center-of-mass movement in the mediolateral direction. When the robot assistance was applied as the center of mass moved toward the opposite leg, the assistance replaced some of the efforts that would have otherwise been provided by the human. This indicates that hip abduction assistance can reduce physical effort during human walking while influencing balance.

Urban dust particles disrupt mitotic progression by dysregulating Aurora kinase B-related functions.

Particulate matter (PM), a major component of outdoor air pollution, damages DNA and increases the risk of cancer. Although the harmful effects of PM at the genomic level are known, the detailed mechanism by which PM affects chromosomal stability remains unclear. In this study, we investigated the novel effects of PM on mitotic progression and identified the underlying mechanisms. Gene set enrichment analysis of lung cancer patients residing in countries with high PM concentrations revealed the downregulation of genes associated with mitosis and mitotic structures. We also showed that exposure of lung cancer cells in vitro to urban dust particles (UDPs) inhibits cell proliferation through a prolonged M phase. The mitotic spindles in UDP-treated cells were hyperstabilized, and the number of centrioles increased. The rate of ingression of the cleavage furrow and actin clearance from the polar cortex was reduced significantly. The defects in mitotic progression were attributed to inactivation of Aurora B at kinetochore during early mitosis, and spindle midzone and midbody during late mitosis. While previous studies demonstrated possible links between PM and mitosis, they did not specifically identify the dysregulation of spatiotemporal dynamics of mitotic proteins and structures (e.g., microtubules, centrosomes, cleavage furrow, and equatorial and polar cortex), which results in the accumulation of chromosomal instability, ultimately contributing to carcinogenicity. The data highlight the novel scientific problem of PM-induced mitotic disruption. Additionally, we introduce a practical visual method for assessing the genotoxic outcomes of airborne pollutants, which has implications for future environmental and public health research.

CCAR2 controls mitotic progression through spatiotemporal regulation of Aurora B.

CCAR2 (cell cycle and apoptosis regulator 2) is a multifaceted protein involved in cell survival and death following cytotoxic stress. However, little is known about the physiological functions of CCAR2 in regulating cell proliferation in the absence of external stimuli. The present study shows that CCAR2-deficient cells possess multilobulated nuclei, suggesting a defect in cell division. In particular, the duration of mitotic phase was perturbed. This disturbance of mitotic progression resulted from premature loss of cohesion with the centromere, and inactivation of the spindle assembly checkpoint during prometaphase and metaphase. It resulted in the formation of lagging chromosomes during anaphase, leading ultimately to the activation of the abscission checkpoint to halt cytokinesis. The CCAR2-dependent mitotic progression was related to spatiotemporal regulation of active Aurora B. In conclusion, the results suggest that CCAR2 governs mitotic events, including proper chromosome segregation and cytokinetic division, to maintain chromosomal stability.

Lighter and Simpler Design Paradigm for Widespread Use of Ankle Exosuits Based on Bio-Inspired Patterns.

Soft wearable robots are attracting immense attention owing to their high usability and wearability. In particular, studies on soft exosuits have achieved remarkable progress. Walking is one of the most basic human actions in daily life. During walking, the ankle joint has considerable influence. Therefore, an exosuit design paradigm having a light and simple structure was developed with the goal of fabricating a soft exosuit that supports the ankle. The new exosuit matches the performance of existing exosuits while being as comfortable as everyday wear. A walking test through a combination with a mobile actuator system, which can maximize these advantages, was also conducted. The combination with the mobile system demonstrates the potential of using the new ankle exosuit as inner wear that maximizes the advantages of a lighter and simpler design. The exosuit design paradigm could serve as an effective guideline for manufacturing assistive exosuits for various body parts in the future.

Histopathology and surgical outcome of symptomatic treatment-related changes after gamma knife radiosurgery in patients with brain metastases.

A late-onset treatment-related changes (TRCs), which represent radiographic radiation necrosis (RN), frequently occur after stereotactic radiosurgery (SRS) for brain metastases and often need surgical treatment. This study aimed to validate the true pathology and investigate clinical implication of surgically resected TRCs on advanced magnetic resonance imaging (MRI). Retrospective analyses of 86 patients who underwent surgical resection after radiosurgery of brain metastases were performed. Fifty-four patients displayed TRCs on preoperative MRI, comprising pure RN in 19 patients (TRC-RN group) and mixed viable tumor cells in 35 patients (TRC-PD group). Thirty-two patients revealed the consistent diagnosis of progressive disease in both MRI and histopathology (PD-PD group). The TRC-PD group showed larger prescription isodose volume (9.4 cm3) than the TRC-RN (4.06 cm3, p = 0.014) group and a shorter time interval from SRS to preoperative MRI diagnosis (median 4.07 months) than the PD-PD group (median 8.77 months, p = 0.004). Progression-free survival was significantly different among the three groups (p < 0.001), but not between TRC-RN and TRC-PD (post hoc test, p = 1.00), while no difference was observed in overall survival (p = 0.067). Brain metastases featured as TRCs after SRS frequently contained viable tumor cells. However, this histologic heterogeneity had a minor impact on benign local prognosis of TRCs after surgical resection.

Direct cardiac rupture associated with sternal fracture following blunt trauma: A report of two case.

Traumatic cardiac injury is not rare. Especially cardiac contusion with sternal fracture due to blunt trauma in common. But cardiac rupture due to direct injury from fractured sternum in very rare. There were two case of cardiac injury supposed to be due to direct injury from fractured sternum. We operated immediately, so we could save these patients. Our cases show that it's rare but blunt trauma could make sternum fracture with direct injury to right side heart.

Expandability of Cephalic Veins after Brachial Plexus Block in Arteriovenous Fistula Formation for Hemodialysis.

BACKGROUND: Arteriovenous fistula (AVF) for hemodialysis is essential for patients with end-stage renal disease. However, it is difficult to maintain AVF reliably. It is vitally important to select proper blood vessels for AVF formation. In a previous study, a minimum diameter of 3 mm for the autologous vein was proposed. However, patients who did not meet the minimum vascular diameter before anesthesia, but fulfilled other criteria, showed satisfactory venous dilatation after brachial plexus block (BPB). This study investigated the extent of vein expansion by BPB and the surgical outcomes of dilated veins after BPB. METHODS: Sixty-one patients who underwent AVF formation using an autologous vein between August 2018 and December 2019 were included in the study. The clinical characteristics of the patient groups, hemodynamic parameters including the diameter of blood vessels before and after BPB, and complications were investigated. Based on the venous diameter measured by sonography before anesthesia, patients were divided into group A (26 patients) and group B (35 patients), with venous diameters <3 mm and ≥3 mm, respectively. RESULTS: The venous diameter expanded after anesthesia by 41% overall, by 62% in group A, and by 25% in group B. This difference between groups A and B was statistically significant (p=0.001). No other variables showed statistically significant differences. CONCLUSION: Sufficient venous dilatation was observed after BPB. Therefore, if the vein is sufficiently dilated after BPB, even in patients with a pre-anesthesia venous diameter <3 mm, surgery may still be performed with an expected desirable outcome.

Localized Pretibial Varicose Vein Caused by an Intraosseous Venous Anomaly.

A 36-year-old man presented to the hospital with protruding blood vessels in his left lower leg accompanied by cramping. An ultrasonographic examination of the leg revealed focal reflux without truncal vein reflux. During phlebectomy, the varix was found to be connected to the intraosseous vein through a tibial opening. Postoperative computed tomography and magnetic resonance imaging showed an osteolytic lesion in the tibial shaft and an intraosseous vascular anomaly. The patient was discharged without complications and scheduled for periodic follow-ups. This young man's varicose vein seemed to be from a tibial intraosseous vascular anomaly, which is extremely rare.

Resuscitative endovascular balloon occlusion of the aorta for retroperitoneal hemorrhage and shock after ipsilateral antegrade angioplasty with vascular closure device.

Percutaneous intervention is widely used to treat peripheral vascular disease. Ipsilateral antegrade femoral arterial access for femoropopliteal disease provides a mechanical advantage with regard to wire and stent control; however, it is associated with vascular complications and significant morbidity and mortality secondary to retroperitoneal hemorrhage from a high puncture site or vascular closure device (VCD) failure. Currently, resuscitative endovascular balloon occlusion of the aorta (REBOA) is performed as damage control surgery in patients with non-compressible torso hemorrhage. We describe a patient with hemorrhagic shock secondary to VCD failure, who was successfully treated by REBOA as damage control surgery. To our knowledge, this is the first reported case in the English literature of successful REBOA in a patient with hemorrhagic shock secondary to VCD failure.

Clinical Outcomes of Arteriovenous Graft in End-Stage Renal Disease Patients with an Unsuitable Cephalic Vein for Hemodialysis Access.

BACKGROUND: As the population of patients with end-stage renal disease has grown older, the proportion of patients with poorly preserved vasculature has concomitantly increased. Thus, arteriovenous grafts (AVG) have been used more frequently to access blood vessels for hemodialysis. Despite this increasing demand, studies of AVG are limited. In this study, we examined the surgical outcomes of upper-limb AVG creation. METHODS: Among the arteriovenous fistula formation procedures performed between January 2014 and March 2019 at Dankook University Hospital, 42 cases involved AVG creation. We compared patients in whom the axillary vein was used (group A; brachioaxillary AVG [B-Ax AVG]; n=20) with those in whom upper limb veins were used (group B; brachiobasilic AVG or brachioantecubital AVG; n=22). RESULTS: The 1-year primary patency rate was higher in group A than in group B (57.9% vs. 41.7%; p=0.262). The incidence of postoperative complications was not significantly different between groups. CONCLUSION: AVG using the axillary vein showed no major differences in safety or functionality compared to AVG using other veins. Therefore, accounting for age, underlying disease, and expected patient lifespan, B-Ax AVG can be considered an acceptable surgical method.